IntroductionIn dental clinic environment dentistry professionals and patients are daily exposed to a great variety of infectious agents and toxic substances transported by aerosols and droplets, produced during dental operative procedures,1promoting an increased risk of cross infection.2,3Aerosols are particles small enough to stay airborne for an extended period before they settle on environmental surfaces.4,5As 75% of these particles drop on a desktop with a diameter of 2m (meters) from the patient position,6the environment (water, air and surfaces) play an important role in this context.2Mouth fluids are grossly contaminated with bacteria, mostly aerobic bacteria (streptococciandstaphylococci) and viruses.2,7Most dental procedures that use handpieces, turbines, ultrasonic scalers, air polishers and abrasion units removes material from the operative site, that becomes aerosolized3,7,8by the instrument rotary action or the water sprays and compressed air combined actions1; So, there is a strong possibility that aerosols, besides the presence of bacteria,9will include viruses, blood, and supra- and sub-gingival plaque organisms.10,11Several studies12,13,14,15,16have been conducted to determine which dental procedure produces the highest airborne bacterial contamination using nonselective bacterial growth media (blood agar). When an aerobic bacterium settles on the plate and grows as a colony, it is counted as a colony-forming unit, or CFU.1Although this method does not provide any differentiation between whether the bacteria are relatively benign or a pathogenic species, it is a sterile, easy to implement and low cost, reproducing the most common conditions and gives a good perspective regarding total airborne bacterial CFUs from a particular procedure.17Pasquarella et al.17described the Index of Air Microbial contamination (IMA) based on the count of the microbial fallout on to Petri dishes left open to the air according to the 1/1/1 scheme (for 1h, 1m from the floor, at least 1m away from walls or any obstacle). Classes of contamination and maximum acceptable levels have been established, and a threshold of 25 was considered adequate.2,3IMA has been tested in many different places and proved to be a reliable and useful tool for monitoring the microbial surface contamination settling from the air in any environment2,17,18although its use is not consensual for critical environments such as operating theatres.2A few studies on air quality microbial counts in dental clinics were presented in literature.2,8,19The aim of this study was to assess the aerosols contamination, by quantifying the aerobic bacteria CFUs when different dental treatments were performed and to analyze the effect of type and time of treatment and time of turbine use on the quantitative bacterial variation of aerosols produced during restorative dentistry and endodontic treatments, and to identify the representative aerobic bacterial colonies. Another purpose of this study was to check the dental clinic atmosphere quality comparing with the IMA values.

Material and methodsThis study was conducted in the teaching Dental Clinical of the Faculty of Health Sciences, University Fernando Pessoa (FHS-UFP). This clinic has a total area of 390m2, divided into 37 dental units spaces (DUs), each one with 5m2. The ventilation system was checked periodically and was not altered during the study period.A baseline register of CFU counts was conducted before any appointment/dental procedure performed in the dental clinic. This control group was constituted by 12 opened plaques placed in three randomly selected DUs. IMA was assessed by passive sampling, using blood agar plates (bioMerieux Ref. 43041, Linda-a-Velha, Portugal) 9cm (centimetre) in diameter that were exposed to air during dental treatments (two at 0.5m and two at 2m from the patient head position) (Figure 1). The plates were opened at the beginning of each dental procedure and remained so for at least 1h to a maximum of 4h (duration of the dental clinical appointments). Overall, 244 air samples in blood agar plates were collected from 26 DUs of the dental clinic.

Figure 1. Representation of the blood agar plates placement regarding the patient head position, on the dental unit space (5m2area) during the operative procedures: (1) box; (2) dental unit; (3) patient head; (4) blood agar plates.

The restorative dentistry procedures included cavities preparation of dental hard tissues disorders that were directly restored with adhesive or non-adhesive dental materials. All biomechanical preparation was done with manual instruments and high-speed handpieces (turbine) with water-cooling. The restored surfaces were polished by rotary action, with water-cooling; Relative operatory field was done with cotton rolls and surgical aspirator.All endodontic treatments, performed in mono- and multi-canal teeth, were non-surgical and included biomechanical preparation with turbine (to prepare the access cavity) with water-cooling, manual instrumentation, disinfection with antimicrobial rinses and the root canal filling; Rubber dam was used for operatory field isolation.To perform the microbial quantitative analysis of air all blood agar plates were incubated at 37C for 48h. Colonies were counted by CFU/plate. The CFU/dm2/h was calculated to determine the IMA. Bacterial levels 2h), use of turbine (yes/no) and time of turbine use (30min/>30min) was performed using the MannWhitney test (as the assumption of normality of the observations does neither hold nor the symmetry of the distributions). The Wilcoxon test was used for comparing median CFU values projected at two different distances (paired measurements, at 0.5m and 2m).

ResultsThe IMA value in the dental clinic was significantly (MannWhitney test,p30min1414.5 (9.5)11.8 (6.023.6)

Different letters (a and b) after the median value stand for significant differences with duration of treatment according to the MannWhitney test.a Blood agar plates placement regarding the patient head position on the dental unit space (5m2area); distance in meters (m).b MannWhitney test.c Due to low n in the no category,p-values for these comparisons were not calculated or were calculated for the two other categories (out of three).* Statistical significant difference.Gram-positive cocci were predominant in the samples:Micrococcussp. (99.9%),Staphylococcus capitis(99.8%),Streptococcussp. (99.9%),Staphylococcus epidermidis(84.8%) andStaphylococcussp. (99.9%) colonies were identified (% of identification reliability given by the API system).

DiscussionIn the present study the air quality of the dental clinic was found to be good, with acceptable values for the air microbial counts17,20since the bacteria levels (11.9 (6.7)CFU/dm2/h) registered were lower than 39CFU/dm2/h.2,17Passive air sampling is one effective way of quantifying airborne bacteria once it measures the live microorganisms that can settle, growth and multiply, as used in the present study, reflects the extent of aerobic bacteria contamination on the surfaces, and highlights clinical areas of primary importance for cross infection prevention.2,8,17The results (Table 1) showed higher number of CFU counts during the endodontic treatments in which the rubber dam was used. One likely explanation is that during the restorative dentistry procedures, the operatory field was relatively isolated with cotton rolls and splatter aspiration. Although the use of rubber dam has been shown to be highly significant in reducing contamination of the atmosphere,8,23another data reported suggest that the use of rubber dam can concentrate and spread24the aerosols produced when high-speed rotating instruments are used or when endodontic treatment is performed, and momentarily contaminates the air; but as the bacteria settle on the surfaces, then the air quality increases. How far the aerosols spread and what level of contamination they cause in the dental surgery is of concern8and should be analyzed.During both dentistry and endodontic treatments CFU counts (Table 1) were significantly higher at 0.5m than at 2m and significantly higher in endodontic treatment for both distances, results corroborated by Timmerman et al.,25but not by Rautemaa et al.,8that registered higher CFU/m2/h counts at distances >1.5m than at distances